1. Field of the Invention
The invention relates to Eisenia foetida polypeptides and peptides, particularly to recombinant polypeptides useful in tumour therapy, microbial infection, inflammation or immunology.
The invention also relates to a process for preparing the above-said polypeptides and peptides.
Furthermore, the invention concerns nucleic acids coding for said polypeptides and peptides.
2. State of the Art
Tumour Necrosis Factor a (TNF-α) is a multifunctional cytokine, produced in vertebrates, mainly by activated macrophages. In vitro, it has several biological effects, including killing of transformed cells and antiparasitic effects. Moreover, TNF-α has been shown to have a lectin-like property for the oligosaccharide ligands chitobiose and Man(a1,3)-Man(a1,6)-Man(1, 2). Recently, Lucas and co-workers (3) have mapped the lectin-like domain of TNF-α and have shown that the domain exerts trypanolytic activity on salivarian trypanosomes such as Trypanosoma brucei. The lectin-like activity of TNF-α is functionally involved in interactions with trypanosomes and possibly also with other pathogens.
The prophenoloxidase (proPO) activating system represents an important defense mechanism in a large variety of invertebrates (4, 5). This system is based on the recognition of bacterial antigens such as lipopolysaccharide (LPS), or peptidoglycan and b-1,3-glucan, present as major components of the cell wall of yeasts and fungi (6, 7). Generally, upon the recognition of such carbohydrates proteinases cleave by limited proteolysis inactive proPO to its active state, phenoloxidase (PO). The active enzyme catalyses the o-hydroxylation of monophenols as well as the oxidation of diphenols to quinones which are subsequently polymerised non-enzymatically to melanin. Melanin and its precursors involved in the proPO activating system have cytotoxic and antimicrobial properties and participate in a wide range of other biological activities including phagocytosis or opsonization, encapsulation or nodule formation, degranulation and wound healing (8–11).
The prophenoloxidase activating system has been detected both in protostomian and deuterostomian species. Though proPO activating system is well documented in arthropods, data in other protostomian groups are more scarce. In annelids, melanization reactions and formation of “brown bodies” or nodules have been described in polychaetes and oligochaetes (12–16). However, biochemical detection of PO activity was so far restricted to a few species with rather controversial results. While Smith and Söderhäll (17) failed to detect proPO system in the polychaete Aphrodite aculeata and Arenicola marina, Fischer (18), Valembois et al. (19), and Porchet-Hennerè and Vernet (15) have evidenced PO activity in Lumbricus terrestris, Eisenia foetida andrei and Nereis diversicolor respectively. More recently, using L-DOPA as substrate, a 30 kDa protein responsible for PO activity was identified in the coelomic fluid of Lumbricus rubellus(20). A report showing that the oxidative activity of the coelomic fluid of earthworms towards L-DOPA in vitro is not affected by trypsin but completely blocked by subtilisin reflects the importance of a correct proteolytic digestion as an initial step for inactive proPO activation (19).
Since the factor which recognizes microbial carbohydrates and triggers the proPO system has not yet been described in annelids (4, 5), investigations were initiated to identify such a molecule in the coelomic fluid (CF) of E. Foetida. Surprisingly, it is shown in this invention that a previously described 42 kDa cytolytic protein named CCF-1 (Coelomic Cytolytic Factor 1) (21) binds LPS and b-1,3 glucan and that the same protein is also responsible for the trypanolytic activity of the coelomic fluid. By combining the glucan and LPS binding capacity with the cytolytic and trypanolytic activity, the invertebrate factor resembles the vertebrate compound TNF-α and can therefore be considered as a primitive type of cytokine, which may be useful as an alternative for TNF-α. This idea is supported by the fact that an anti-TNF monoclonal antibody (anti-TNF/TIP) crossreacts with CCF-1, whereas an anti-CCF-1 monoclonal antibody (12C9) crossreacts with TNF-α. Moreover, in E. foetida, CCF-1 levels are increased after LPS treatment, which ressembles the TNF induction by LPS, noticed in vertebrates. Apart from the above described characteristics, it is shown that CCF-1 also participates in the proPO cascade of the coelomic fluid of Eisenia foetida.
Even more surprisingly, the cytolytic, trypanolytic and glucan-binding characteristics of the protein can be attributed to a small domain of 13 amino acids as depicted in SEQ ID NO:1. Moreover, this isolated peptide of 13 amino acids shows biological activity. The sequence of this peptide, however, is completely different from the TIP region of TNF-α, although it shares some functional characteristics.